Sample processing device and method

ABSTRACT

A device for processing a biological sample includes a processing unit having at least one opening to receive a sample vessel and a plurality of processing stations positioned along the opening. The processing stations each have a compression member adapted to compress the sample vessel within the opening and thereby move the sample within the sample vessel among the processing stations. An energy transfer element can be coupled to one or more of the processing stations for transferring thermal energy to the sample at a processing station. The device can be used for PCR processing of nucleic acid samples. A sample vessel of the present invention can be a tubule flow-chamber having a plurality of segments separated by pressure gates. The sample vessel minimizes sample handling by providing a closed tubule in which distinct processing steps can be carried out in each of the segments of the tubule.

REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSerial No. 60/259,025 filed on Dec. 29, 2000. The contents of theaforementioned application are hereby incorporated by reference.

BACKGROUND

As result of the Human Genome Project and other genetic research, atremendous amount of genomic and biomarker information is presentlyavailable to healthcare providers. Using molecular diagnostic testing,genomic and biomarker information can provide a resource to healthcareproviders to assist in the rapid and accurate diagnosis of illness.However, the development of diagnostic testing systems allowing the useof such genetic information, particularly in the clinical setting, hasfailed to match pace with the genetic research providing theinformation. Current diagnostic testing systems are mainly limited tolarge medical testing centers or research labs due to the high costsassociated with acquiring and operating the systems and the complexityof the molecular diagnostic assays being employed. These current systemsrequire a large initial capital investment and incur high costs forreagents, disposables, operation, maintenance, service and training.

SUMMARY

The present invention provides sample processing devices and methodsthat facilitate the rapid analysis of biological samples, such as blood,saliva, or urine, in an efficient and cost effective manner withminimal, if any, exposure to biohazards. The sample processing devicesand methods of the present invention are particularly suited to theclinical setting, allowing the clinician to readily proceed fromacquisition of a test sample to analysis of the test results, withminimal human intervention. The sample processing devices of the presentinvention may be implemented as a hand-held system suitable for theprocessing of a single sample or as a larger, bench top unit suitablefor the simultaneous processing of multiple samples. The presentinvention may be valuable in all diagnostic and therapeutic monitoringareas, including in the point-of-care or clinical setting, inhigh-throughput screening, and in biological warfare detection. Inaddition, the present invention provides a sample vessel for holding abiological sample throughout the processing of the sample.

In accordance with one embodiment of the present invention, a device forprocessing a sample includes a processing unit having an opening toreceive a sample vessel and at least one processing station positionedalong the opening. The processing station includes a compression memberadapted to compress the sample vessel within the opening and therebydisplace a content of the sample vessel within the sample vessel. Thecontent displaced by the compression member can be, for example, thesample, a reagent, or a mixture of the content and a reagent.

In accordance with another aspect, the processing station may include anenergy transfer element for transferring energy to or from the contentwithin the sample vessel and a control system coupled to the energytransfer element to control the energy transferred to or from thecontent. The energy transfer element can be, for example, an electronicheat element, a microwave source, a light source, an ultrasonic sourceor a cooling element.

In accordance with a further aspect, the energy transfer elementtransfers thermal energy to or from the content within the samplevessel. An energy insulator may be positioned adjacent the processingstation. The energy insulator can be, for example, an energy shieldinglayer, an energy absorption layer, an energy refraction layer, or athermal insulator, depending on the type of energy transfer elementemployed. A temperature sensor may be coupled to the control system tomonitor temperature at the processing station. Alternatively, theprocessing station may include a heat sink to dissipate thermal energyfrom the processing station.

In accordance with another aspect, the processing station may include astationary member opposing the compression member across the opening.The compression member can operate to compress the sample vessel againstthe stationary member within the opening.

In accordance with a further aspect, a driver may be coupled to thecompression member to selectively move the compression member andthereby compress the sample vessel within the opening. The driver canbe, for example, a motor coupled to the compression member by a cam.Alternatively, the driver can be an electromagnetic actuating mechanism.

In accordance with another aspect, the processing device can include asensor for detecting a signal from the content within the sample vessel.An energy source can optionally be provided for applying energy to thecontent within the sample vessel to generate a signal from the content.In one embodiment, the processing device can include an electrophoresissystem comprising a pair of electrodes adapted to have a predeterminedvoltage difference and an electrode actuator for inserting theelectrodes into the sample vessel.

In accordance with a further aspect, the processing device may include areagent injector cartridge actuator adapted to receive a reagentinjector cartridge having at least one needle in fluid communicationwith a reagent reservoir. The reagent injector cartridge actuator can beoperable to move the reagent injector cartridge to inject a quantity ofreagent into the sample vessel.

In accordance with another embodiment of the invention, a sample vesselfor holding a sample includes a sample containing portion for holdingthe sample and a handling portion for handling the sample vessel. Thesample containing portion can have a wall constructed of a flexiblematerial permitting substantial flattening of a selected segment of thesample containing portion. The handling portion can be coupled to thesample containing portion and preferably has a generally rigidconstruction to facilitate handling of the sample vessel.

In accordance with another aspect, the sample containing portion of thesample vessel can be a tubule.

In accordance with a further aspect, the sample vessel can include atleast one pressure gate disposed within the sample containing portion todivide the sample containing portion into a plurality of segments. Atleast one of the segments of the sample vessel can have a filtercontained therein that is structured to separate selected components ofa sample material from other components of the sample material.Additionally, at least one of the segments of the sample vessel cancontain a reagent. The reagent can be, for example, an anticoagulant, acell lyses reagent, a nucleotide, an enzyme, a DNA polymerase, atemplate DNA, an oligonucleotide, a primer, an antigen, an antibody, adye, a marker, a molecular probe, a buffer, or a detection material. Thesample containing portion also can include an electrophoresis segmentcontaining a gel for electrophoresis. The electrophoresis segment caninclude a pair of electrodes adapted to maintain a predetermined voltagedifference therebetween. Additionally, one of the segments can containmultilayer membranes or a micro-array bio-chip for analyzing the sample.

In accordance with another aspect, the sample containing portion caninclude a self-sealing injection channel formed therein. The selfsealing injection channel is preferably normally substantially free ofsample material and capable of fluid communication with the samplematerial in the sample containing portion.

In accordance with another aspect, the sample vessel can include aninstrument for obtaining a sample coupled to the sample vessel.

In accordance with a further aspect, the handling portion of the samplevessel includes an opening for receiving a sample. The sample vesselalso can include a closure for selective closing the opening.Preferably, the closure seats against the handling portion to close theopening. In addition, the instrument for obtaining a sample can becoupled to the closure of the sample vessel.

In accordance with another aspect, the handling portion has a wallthickness greater than a thickness of the wall of the sample containingportion. Preferably, the thickness of the wall of the sample containingportion is less than or equal to 0.3 mm. In one embodiment, the handlingportion can include a cylindrical sleeve sized and shaped to fit over aportion of the sample containing portion. The handling portion ispreferably positioned longitudinally adjacent the sample containingportion.

In accordance with another embodiment, a sample vessel for holding asample includes a sample containing portion having at least one pressuregate disposed within the sample containing portion to divide the samplecontaining portion into a plurality of segments. Preferably, at leastone segment of the sample containing portion has a wall constructed of aflexible material permitting substantial flattening of the segment ofthe sample containing portion.

In accordance with another embodiment, a method of processing a samplewithin a sample vessel includes the steps of introducing the samplevessel into a device for processing the sample and compressing thesample vessel to move the sample within the sample vessel from a firstsegment to a second segment of the sample vessel.

In accordance with another aspect, the method of processing a sample caninclude the step of introducing a reagent to the sample within a segmentof the sample vessel.

In accordance with a further aspect, the method of processing a samplecan include the step of heating the sample in the first segment to afirst temperature. The method can also include the step of heating thesample to a second temperature in the second segment. In one embodiment,the first temperature can be effective to denature the sample and thesecond temperature is one at which nucleic acid annealing and nucleicacid synthesis can occur. The method of processing a sample can furtherinclude the steps of compressing the sample vessel to move the samplewithin the sample vessel from the second segment to the first segment ofthe sample vessel and heating the sample to the first temperature in thefirst segment.

In accordance with another aspect, the method of processing the samplecan include the step of analyzing the sample by detecting a signal fromthe sample within a segment of the sample vessel and analyzing thedetected signal to determine a condition of the sample. The analyzingstep can include applying an excitation energy to the sample within thesegment of the sample vessel. Additionally, the analyzing step caninclude conducting electrophoresis analysis of the sample by applying aselective voltage to the sample within a segment of the sample vessel,detecting light emitted from the sample, and analyzing the detectedlight to determine a condition of the sample.

Alternatively, the analyzing step can include applying an excitationenergy to a bio-array member contained within a segment of the samplevessel, detecting light emitted from the bio-array member, and analyzingthe detected light to determine a condition of the sample. The bio-arraymember can be, for example, a multi-layer membrane or a micro-arraybio-chip.

In accordance with a further aspect, the method of processing a samplecan include the step of agitating the sample within a segment of thesample vessel.

In accordance with another embodiment, a method of treating a samplewithin a sample vessel can include the steps of introducing the samplevessel into a device for processing the sample within the sample vesseland compressing one of the segments to mix the reagent with the samplewithin the sample vessel. Preferably, the sample vessel has a pluralityof segments including a segment for containing a reagent and a segmentfor containing the sample.

In accordance with another aspect, the method of processing the samplecan include the step of introducing the reagent into a reagent segmentof the sample after the step of introducing the sample vessel into thedevice for processing the sample.

In accordance with another embodiment, a thermal cycler includes aprocessing unit having an opening to receive a sample vessel containinga sample. The processing unit can have a first processing station, asecond processing station, and a third processing station positionedalong the opening. The first processing station can include a firstcompression member adapted to compress the sample vessel within theopening and a first energy transfer element for transferring energy tothe sample at the first processing station. The second processingstation can include a second compression member adapted to compress thesample vessel within the opening and a second energy transfer elementfor transferring energy to the sample at the second processing station.The third processing station can include a third compression memberadapted to compress the sample vessel within the opening and a thirdenergy transfer element for transferring energy to the sample at thethird processing station. Compression of the sample vessel by of one ofthe compression members can displace the sample within the sample vesselbetween the processing stations.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bemore fully understood by reference to the following detailed descriptionin conjunction with the attached drawings in which like referencenumerals refer to like elements through the different views. Thedrawings illustrate principles of the invention and, although not toscale, show relative dimensions.

FIG. 1 is a schematic diagram of a device for processing a sampleaccording to the present invention;

FIG. 2 is a schematic diagram of the device of FIG. 1, illustrating acompression member of a processing station of the device compressing thesample vessel;

FIG. 3 is a schematic diagram of an alternative embodiment of a devicefor processing a sample according to the present invention;

FIG. 4 is a schematic diagram of an alternative embodiment of a devicefor processing a sample according to the present invention;

FIG. 5 is a perspective view of an embodiment of a hand held device forprocessing a sample according to the present invention;

FIG. 6 is a perspective view of an embodiment of a bench top device forprocessing a sample according to the present invention;

FIG. 7 is a perspective view of the device of FIG. 6, illustrating thedevice with the top cover removed;

FIG. 8 is a perspective view of an embodiment of a thermal cyclingprocessing unit according to the present invention;

FIG. 9 is a perspective view of the processing unit of FIG. 8;

FIG. 10 is a partially exploded, perspective view of a processingstation of the processing unit of FIG. 8, illustrating a heat block unitand an insulator block unit of the processing station;

FIG. 11 is a partially exploded, perspective view of the processing unitof FIG. 8, illustrating a plurality of heating block units and insulatorblock units;

FIG. 12 is a partially exploded, perspective view of a processingstation of an alternative embodiment of a processing unit according tothe present invention;

FIGS. 13A-13G are side elevational views, in cross-section, of aprocessing unit of the present invention, illustrating the operation ofthe processing unit;

FIG. 14 is a side elevational view, in cross section, of a gelelectrophoresis analysis unit of the present invention;

FIGS. 15A-15B are side elevational views, in cross-section, ofembodiments of a sample vessel according to the present invention;

FIG. 16 is a side elevation view, in cross section, of a portion of asample vessel according to the present invention, illustrating aninjection channel formed in the sample vessel;

FIG. 17 is a side elevational view of a reagent cartridge according tothe present invention;

FIG. 18 is a side elevational view, in cross-section, of a sample vesselaccording to the present invention; and

FIGS. 19A-19C illustrate an alternative embodiment of a processing unitof the present invention.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present invention provides devices and methods for processing asample. The term “processing” as used herein generally refers to thepreparation, treatment, analysis, and/or the performance of othertesting protocols or assays on a content of the sample vessel in one ormore steps. Exemplary processing steps include, for example: displacinga content, e.g., the sample or a reagent, of the sample vessel withinthe sample vessel to, for example, adjust the volume of the content,separate content components, mix contents within the sample vessel;effecting a chemical or biological reaction within a segment of thesample vessel by, for example, introducing a reagent to the sample,agitating the sample, transferring thermal energy to or from the sample,incubating the sample at a specified temperature, amplifying componentsof the sample, separating and/or isolating components of the sample; oranalyzing the sample to determine a characteristic of the sample, suchas, for example, the quantity, volume, mass, concentration, sequence, ornucleic acid size or other analyte size, of the sample. One skilled inthe art will appreciate that the forgoing exemplary processing steps aredescribed herein for illustrative purposes only. Other processing stepsmay be employed without departing from the scope of the presentinvention.

A device for processing a sample according to the present invention canintegrate one or more processing units into a single system depending onthe process being employed. The processing units can include one or moreprocessing stations at which one or more processing steps can beperformed on the sample within the sample vessel. Sample materials thatcan be processed according to the present invention are generallybiological samples or samples containing biological substance andinclude, for example, blood, urine, saliva, cell suspensions, biofluids,a piece of tissue, soil or other samples. A sample processing device ofthe present invention is particularly suited for nucleic acidamplification, such as polymer chain reaction (PCR) or ligase chainreaction (LCR) amplification, and can include, for example, a samplepretreatment unit for extracting nucleic acid from sample, a thermalcycling reaction unit for amplification of the nucleic acid or signal,and (optionally) an analysis or detection unit for analyzing theamplified product. The sample processing device of the present inventioncan also be used for isothermal reaction of nucleic acid or signalamplifications, such as strand displacement amplification (SDA), rollingcircle amplification (RCA), and transcription-mediated amplification(TMA). Other exemplary processes to be performed on samples can includeclinical diagnosis, therapeutic monitoring, and screening of chemicalcompounds for discovery of new drugs. The following descriptionprimarily focuses on PCR amplification for illustration. However, oneskilled in the art will appreciate that the devices and methods of thepresent invention are not limited to PCR amplification, as the devicesand methods described below can be employed in other sample processing.

An exemplary embodiment of a device for processing a sample isillustrated in FIG. 1. The processing device 10 illustrated in FIG. 1includes a processing unit 12 having an opening 14 to receive a samplevessel 16. The opening 14 can be a tubular shaped opening, an open-facedslot or other structure for receiving the sample vessel 16 in aremovable and replaceable manner. The processing unit 12 includes afirst processing station 18 and a second processing station 20, eachpositioned along the length of the opening 14. The first processingstation 18 includes a compression member 22 adapted to compress thesample vessel 16 within the opening 14 and thereby displace a content ofthe sample vessel within the sample vessel 16. The content of the samplevessel can be, for example, the sample, a reagent contained within thesample vessel, or a mixture of the sample and the reagent. A driver 24is coupled to the compression member 22 to selectively move thecompression member 22 and thereby compress the sample vessel 16 withinthe opening 14. The driver 24 can be, for example, an electromagneticactuating mechanism, a motor, a solenoid, or any other device forimparting motion, preferably reciprocal motion, to the compressionmember 22, as described in further detail below.

Preferably, the compression member 22 is constructed from a rigidmaterial such as a rigid plastic or a metal. The compression member canbe constructed in any shape sufficient to impart a compressive force onthe sample vessel. For example, the compression member 22 can be a blockhaving a rectilinear, planar surface for engaging the sample vessel 16,as illustrated in FIG. 1. Alternatively, the compression member can havea curved, angular, or non-planar surface for engaging the sample vessel16.

Moreover, the compression member 22 alternatively can be an inflatablemembrane that can be inflated by a fluid, e.g., air, nitrogen, saline,or water, to impart a compressive force on the sample vessel. In thisembodiment, the amount of compression of the sample vessel may becontrolled by the adjusting the inflation pressure of the membrane.

The first processing station 18 can optionally include a stationarymember 26 positioned opposite the compression member 22 across theopening 14. The compression member 22, thus, can compress a portion ofthe sample vessel 16 within the opening 14 against the stationary member26, as illustrated in FIG. 2. One skilled in the art will appreciatethat the stationary member 26 may be replaced with a second compressionmember, such that the processing station includes two compressionmembers that move together to compress the sample vessel therebetween.In addition, a stationary member or second compression member may beomitted by securing the sample vessel 16 within the opening on eitherside of the compression member.

In the illustrated embodiment, the sample vessel 16 is a closed tubuleflow-chamber for holding the sample. Preferably, one or more segments ofthe sample vessel 16 are constructed of a flexible, compressiblematerial, such as, for example, polyethylene or polyurethane, to allowselective compression, and preferably flattening, of the sample vesselto move the sample, or other contents of the sample vessel, within thesample vessel, preferably while the sample vessel 16 remains in thedevice 10. In one preferred embodiment, the sample vessel 16 includes aplurality of segments separated by an integral, internal structure, suchas a micro-fluidic pressure gate, as described in more detail below.Alternatively, the sample vessel 16 can be constructed without internal,integral structures to form segments and the device 10 can be utilizedto segment the sample vessel by compressing selective portions of thesample vessel. One skilled in the art will appreciate that other typesof sample vessels suitable for containing a sample may be used with thedevice 10 without departing from the scope of the present invention.

The second processing station 20 can include a sensor 28 for detecting asignal from the content, e.g., the sample or a reagent, of the samplevessel 16. For example, the sensor 28 can be an optical sensor formeasuring light, for example fluorescent light, emitted from the sampleor from fluorescent probes within the sample. In addition, multiplesensors or a spectrum sensor can be used when detection of multiplewavelength light is required. The detected signal can be sent to a CPU30 to analyze the detected signal and determine a characteristic of thesample.

In operation, a sample can be introduced to a first segment A of thesample vessel 16 by injecting the sample through the walls of the samplevessel 16 or by introducing the sample through an opening formed in thesample vessel 16, as described in more detail below. In the presentexemplary embodiment illustrated in FIGS. 1 and 2, the sample vessel 16includes a pressure gate 32 that divides the sample vessel 16 into afirst segment A and a second segment B. The sample vessel 14 can beinserted into the opening 14 of the device 10 such that the firstsegment A of the sample vessel 16 is aligned with the first processingstation 18 and the second segment B is aligned with the secondprocessing station 20, as illustrated in FIG. 1.

The driver 24 can operate to move the compression member 22 into contactwith the sample vessel 16 such that the first segment A of the samplevessel 16 is compressed within the opening 14 between the compressionmember 22 and the stationary member 26. As the first segment A of thesample vessel 16 is compressed, a quantity of sample is displaced fromthe first segment A to the second segment B through the pressure gate32. The volume of sample displaced is proportional to the amount ofcompression of the first segment A by the compression member 22. Thus,the compression member 22 of the first processing station 18 can be usedto displace a specific quantity of sample into the second segment B ofthe sample vessel 16 for analysis at the second processing station 20.Substantially all of the sample can be displaced from the first segmentA of the sample vessel 16 by completely flattening the first segment Aof the sample vessel 16, as illustrated in FIG. 2. The sample can beanalyzed in the second segment B of the sample vessel 16 at the secondprocessing station 20.

An alternative embodiment of a device for processing a sample isillustrated in FIG. 3. The device 38 includes a processing unit 40having three processing stations positioned along the opening 14,namely, a first process station 42, a second processing station 44adjacent the first processing station 42, and a third processing station46 adjacent the second processing station 44.

The first processing station 42 includes a compression member 22 coupledto a driver 24 and adapted to compress a segment of the sample vessel 16against a stationary member 26 within the opening 16. The firstprocessing station 42 can operate to displace a selective quantity ofthe sample from a first segment A of the sample vessel into othersegments of the sample vessel.

The second processing station 44 includes a compression member 22coupled to a driver 24 and adapted to compress a second segment B of thesample vessel 16 against a stationary member 26 within the opening 16.The second processing station 44 includes an energy transfer element 48for transferring energy to or from the contents of the sample vessel 16.The energy transfer element 48 can be, for example, an electronic heatelement, a microwave source, a light source, an ultrasonic source, acooling element, or any other device for transferring energy. In oneembodiment, the energy transfer element 48 transfers thermal energy toor from the sample within the sample vessel. The energy transfer element48 can be embedded in or otherwise coupled to the compression member 22,as illustrated in FIG. 3. Alternatively, the energy transfer element 48can be coupled to the stationary member 26 or can be positioned withinthe processing station independent of the compression member or thestationary member. The energy transfer element 48 can be coupled to acontrol system that controls the energy transferred to or from thesample vessel 16 by the energy transfer element 48. The control systemcan be a component system of the CPU 30 or can be an independent system.The control system can also include a temperature sensor 50 to monitorthe temperature of the energy transfer element.

The second processing station 44 also can include a sensor 52 fordetecting a signal from the content of the sample vessel, particularlyduring processing in the second processing station. For example, thesensor 52 can be an optical sensor for measuring light, for examplefluorescent light, emitted from the sample or from fluorescent probeswithin the sample. The sensor 52 can be coupled to the CPU 30 foranalysis of the detected signal to determine a characteristic of thesample.

The third processing station 46 can include a sensor 28 for detecting asignal from the content, e.g., the sample or a reagent, of the samplevessel 16. For example, the sensor 28 can be an optical sensor formeasuring light, for example fluorescent light, emitted from the sampleor from fluorescent probes within the sample. In addition, multiplesensors or a spectrum sensor can be used when detection of multiplewavelength light is required. The detected signal can be sent to a CPU30 to analyze the detected signal and determine a characteristic of thesample.

In operation, a sample can be introduced into a first segment A of thesample vessel 16 and the sample vessel 16 can be introduced into theopening 14 of the device 10. In the embodiment illustrated in FIG. 3,the sample vessel 16 includes two pressure gates 32 that divide thesample vessel 16 into three segments, namely, the first segment A, asecond segment B, and a third segment C. The first processing station 42can operate to displace a selective amount of the sample into the secondsegment B of the sample vessel 16 for processing at the secondprocessing station 44.

At the second processing station 44, energy can be transferred to orfrom the sample within the second segment B. In this manner, abiological or chemical reaction involving the sample may be carried outin the second segment B. The sensor 52 can be used to monitor thereaction during the reaction process.

Upon completion of the reaction, the sample can be moved into the thirdsegment C of the sample vessel 16 by compressing the sample vessel 16within the opening at the second processing station 44. Preferably, thecompression member 22 of the first processing station 42 substantiallyflattens the first segment A of the sample vessel 16 to inhibit thesample from entering the first segment A. The sample can be analyzed inthe third segment C of the sample vessel 16 at the third processingstation 46.

A further embodiment of a device for processing a sample is illustratedin FIG. 4. The device 56 includes a processing unit 58 having aprocessing station 60 positioned along the opening 14. The processingstation 60 includes a compression member 22 coupled to a driver 24 andadapted to compress a segment of the sample vessel 16 against astationary member 26 within the opening 16. In the embodimentillustrated in FIG. 4, the sample vessel 16 includes a pressure gate 32that divides the sample vessel 16 into two segments, namely, a firstsegment A and a second segment B. The processing station 60 can operateto displace a selective quantity of the content from the second segmentB of the sample vessel into the first segment A of the sample vessel.For example, a reagent can be introduced into the second segment B ofthe sample vessel 16. A quantity of reagent can be displaced from thesecond segment B into the first segment A of the sample vessel 16 to mixwith the sample in the first segment A. Alternatively, the reagent canbe introduced into the first segment A of the sample vessel 16 and aquantity of the sample can be displaced from the second segment B intothe first segment A by the processing station 60. Thus, the firstsegment A of the sample vessel 16 can act as a reaction mixture chamberfor the sample and the reagent. The reagent can be pre-packaged in thesample vessel 16 or can be introduced to the sample vessel 16 after thesample is introduced to the sample vessel 16. For example, the reagentcan be introduced using a reagent injector cartridge, described below,that is included with the device.

Referring to FIG. 5, another embodiment of device for processing asample is illustrated. The illustrated device 100 is a hand held systemfor processing a nucleic acid sample, preferably in an “insert and test”format in which a sample vessel containing a nucleic acid sample isinserted into the device 100 and processing results are produced by thedevice with minimal human intervention. The device 100 can include ahousing 112 having an opening 114 for receiving a sample vessel 116containing a sample for processing by the device 100. The opening 114can be a tubular shaped opening, as illustrated in FIG. 5, or can be anopen-faced slot or other structure for receiving the sample vessel in aremovable and replaceable manner. A control panel 118 is located on thetop of the housing 112 for inputting information to the device 100 and amonitor 120 is provided for displaying operating information, such asthe results of processing. An external communication port 121 can belocated on the housing 112 for receiving information or outputtinginformation, such as the results of processing and remote diagnosing ofthe system, to a remote system, such as a computer network. A battery123 (FIG. 7) can be located within the housing to provide electricalpower to the components of the device 100.

A multi-sample device 200 for processing multiple samples is illustratedin FIG. 6. The device 200 is a bench top thermal cycling system forprocessing up to 96 nucleic acid samples simultaneously. The sampleprocessing device 200 operates on the same principals as the sampleprocessing device 100 illustrated in FIG. 5, except that themulti-sample device 200 provides increased capacity and throughput. Themulti-sample processing device 200 can include a housing 202 having aplurality of wells or openings 204, with each well being capable ofreceiving a sample vessel 206 containing a sample for processing by thedevice. The exemplary multi-sample device 200 illustrated in FIG. 6 hasninety-six wells for treating up to 96 samples simultaneously. Oneskilled in the art will appreciate that a multi-sample processing deviceaccording to the present invention may be designed with any number ofwells, depending on the sample being tested and the processes beingemployed, without departing from the scope of the present invention. Acontrol panel 208 is located on the top of the housing 202 for inputtinginformation to the multi-sample processing device 200 and a monitor 210is provided for displaying operating information, such as the results oftesting.

FIG. 7 illustrates the general components of the sample processingdevice 100 illustrated in FIG. 5. The illustrated device 100 includesthree primary processing units for processing a sample within the samplevessel, namely, a pretreatment unit 122 for pretreating the sample, areaction unit 124 for amplifying certain components of the sample, andan analysis unit 126 for analyzing the sample. The sample vessel can beloaded into the device 100 through the opening 114. The processing unitsof the device are preferably arranged along the axis of elongation ofthe opening 114. This arrangement allows the sample to be moved withinthe sample vessel between the processing units of the device 100 in amanner described in detail below. Preferably, the processing units arearranged linearly as illustrated in FIG. 7, however, other arrangementare possible so long as the sample vessel can be positioned adjacent oneor more of the processing units of the device 100.

Continuing to refer to FIG. 7, a pair of sample vessel loading wheels128 is located at the entrance 130 of the sample vessel opening 114. Theentrance 130 is preferably tapered to facilitate loading of the samplevessel into the opening 114 of the device 100. The loading wheels 128further facilitate loading of the sample vessel by guiding the samplevessel into the opening 114. A sample collection unit 132 can bepositioned proximate the entrance 130 of the opening 114 to allow aselective volume of the sample to dispense into the next processing unitor units within the sample vessel. The sample collection unit 132 caninclude a compression member 22 opposed to a stationary member 26 acrossthe width of the opening 114. A linear motor 138 is coupled to thecompression member 22. The linear motor 138 can operate to move thecompression member 22 toward or away from the stationary member 26 toselectively open and close the opening 114 therebetween. When the samplevessel is positioned within the opening 114, the linear motor 138 canoperate to compress the sample vessel between the compression member 22and the stationary member 26. As a result, a selective volume of thesample can be moved to the next processing unit within the samplevessel. Preferably, the sample vessel remains compressed between thecompression member 22 and the stationary member 26 of the samplecollection unit 132 during processing of the sample by the otherprocessing units to prevent the sample from exiting the processing unitarea during processing.

The pretreatment unit 122 is positioned adjacent the initial samplecollection unit 132. Depending on the process being implemented, thesample may require pretreatment or preparation before proceeding withadditional processing steps. Pretreatment can include, for example,adding a reagent or other material to the sample and incubating themixture for certain time period. The pretreatment unit 122 of the device100 allows for any of such pretreatment steps to be implemented. For PCRtesting, the sample pretreatment unit 122 can provide for nucleic acidextraction from a biological sample, such as blood. Any known methodsfor extracting nucleic acid can be utilized in the pretreatment unit,including using a cell lysis reagent, boiling the nucleic acid sample,GITC, or formamide for solubilization. Alternatively, filters can beused within the sample vessel to separate nucleic acid from unwantedcellular debris.

The pretreatment unit 122 can include a compression member 22 and astationary member 26 opposed to the compression member 26 across theopening 114. The compression member 22 and/or the stationary member 26can optionally include an energy transfer element for transferringenergy, e.g. thermal energy, to the sample within the sample vessel. Theenergy transfer element can be, for example, an electronic heat element(such as Kapton heater, a Nomex heater, a Mica heater, or a siliconerubber heater), a microwave generator, a light source, an electroniccooling element (such as Peltier element), an ultrasonic energy transferelement, or any another device suitable for transferring thermal energy.A driver 24, for example an electromagnetic actuator such as linearstepper actuator, a relay actuator, or a solenoid, is coupled to thecompression member 22 and operates as a driver. During operation of thepretreatment unit 122, the driver 24, moves the compression member 22 toopen the opening 114 between the compression member 22 and thestationary member 26 of the pretreatment unit 122 to allow receipt of asample vessel. After a sample vessel is loaded, the driver 24 drives thecompression member 22 toward the stationary member 26, resulting in goodsurface contact between the sample vessel and the compression member andthe stationary member and thus improved pretreatment. Once thepretreatment is completed, the driver 24 moves the compression member 22of the pretreatment unit 122 to further compress the pretreatmentsegment of the sample vessel to move a selective amount of pretreatedsample within the sample vessel to the next processing unit.

The reaction unit 124 can include a plurality of processing stations150A-150C and is preferably positioned adjacent the pretreatment unit122. The reaction unit 124 can affect thermal cycling of the sample byselectively moving the sample, with the sample vessel, between theprocessing stations 150A-150C. The phrase “thermal cycling” as usedherein refers to a process of heating and/or cooling a sample in two ormore steps, with each step preferably occurring at a differenttemperature range from the previous step. Each of the processingstations 150A-150C can be maintained at a pre-selected temperature rangecontrolled by a temperature control system 152 and a CPU 174. Althoughthe exemplary embodiment includes three thermal cycling processingstations 150A-150C, the reaction unit 124 can include any number ofprocessing stations 150, depending on the thermal cycling processemployed. Alternatively, the reaction unit 124 can incubate a sample ata selective temperature for an isothermal reaction such as for TMA orSDA process.

In PCR based testing, thermal cycling can be used to denature, anneal,elongate and thereby amplify the nucleic acid sample. The PCR thermalcycling steps each occur at specified temperature ranges. Denaturingoccurs at approximately 92° C.-96° C.; elongation occurs atapproximately 70° C.-76° C.; and annealing occurs at approximately 48°C.-68° C. Each of the PCR thermal cycling steps, i.e. denaturing,annealing, and elongation, can be carried out independently at aseparate processing station of the reaction unit 124 by maintaining theprocessing stations at the temperature ranges effective for carrying outeach of the PCR thermal cycling steps. For example, the denaturing stepcan be carried out at processing station 150A, the elongation step atprocessing station 150B, and the annealing step at processing station150C. Alternatively, one or more of the PCR thermal cycling steps can becombined and carried out at the same processing station, therebyreducing the number of processing stations required. For example,denaturing can be carried out at processing station 150A and elongationand annealing can be carried out at processing station 150B, thus,eliminating the need for a third processing station.

Moreover, a processing station can be provided within the reaction unit122 for cooling of the sample by using a thermal energy element, aPeltier thermal electric element for example, to transfer thermal energyfrom the processing station. In PCR processing, for example, aprocessing station can be provided to preserve the nucleic acid samplebetween process steps by cooling the sample to a refrigerationtemperature, e.g., 4° C. Additionally, a processing station canoptionally be provided to cool the sample between thermal cycling stepsto decrease the temperature down ramping time between successive thermalcycling steps. For example, as denaturing generally occurs at 92° C.-96°C. and annealing generally occurs at a significantly lower temperature,e.g., 48° C.-68° C., the sample can be cooled after denaturing in acooling processing station, preferably at a temperature lower than theannealing temperature, to bring the sample temperature more quickly intothe annealing temperature range. A thermal cycling processing stationcan optionally include a heat sink 166 coupled to either the compressionmember 22 or the stationary member 26 to conduct heat away from thestation and radiate the heat to the environment.

Each of the illustrated processing stations of the reaction unit 124includes a compression member 22 and a stationary member 26. Thecompression member 22 of each thermal cycling processing unit can becoupled to a driver 24 for selectively moving the compression member 22toward and away from the stationary member 26. As discussed above, thedrivers 24 can be any device capable of imparting motion, preferablyreciprocal motion, to the compression members. A driver control system160 is coupled to the drivers 24 to control the operation of the drivers24. In one preferred embodiment illustrated in FIG. 7, the drivers 24are electromagnetic actuators coupled to the driver control system 160,which can be, for example, a control system for controlling thereciprocal motion of the actuators. Alternative drivers, compressionmembers and stationary members are described below in connection withFIGS. 8-12. The driver control system 160 is coupled to the CPU 174 suchthat the sample incubation time period, the pressure and the samplemoving speed within the sample vessel can be controlled and coordinatedby the CPU 174 to achieve the best reaction results.

Each of the thermal cycling processing station 150A-150C can optionallyinclude an energy transfer element for transferring energy, such asthermal energy, to the sample within the sample vessel. The energytransfer elements can be, for example, an electronic heat element, amicrowave generator, a light source, an electronic cooling element, orany another device suitable for applying thermal energy. Each of theenergy transfer elements is coupled to the temperature control system152 to maintain the associated processing station within a selectedtemperature range. One or more temperature sensors, coupled to thetemperature control system 152, can be positioned proximate theprocessing stations 150A-150C to monitor the temperature of thestations.

Between two adjacent processing units or two adjacent processingstations, at least one layer of energy insulator 146 can optionally beprovided to insulate the processing unit or the processing station fromadjacent units or stations. An energy insulator layer can also be formedon the boundary of a processing station to prevent energy transfer to orfrom the environment. The energy insulator 146 can be, for example, anenergy shielding layer, an energy absorption layer, an energy refractionlayer, or a thermal insulator, depending on the type of energy transferelement employed. A thermal insulator can be constructed from a lowthermal conductivity material such as certain ceramics or plastics. Inone embodiment, the thermal insulator can be attached to the compressionmembers and the stationary members. Alternatively, the thermalinsulators can be separate from the compression members and stationarymembers and can be controlled independently by a driver to open andclose the opening 114. In this embodiment, all the compression membersand insulators can open initially to allow loading of the sample vessel,and then, the thermal insulators can compress the sample vessel withinthe opening to close the vessel and form separate segments within thesample vessel. Additionally, a spring element or other biasing mechanismcan be optionally utilized to bias each thermal insulator. Through thespring element, a driver associated with one of the thermal insulatorscan apply sufficient pressure on the thermal insulator to minimize thequantity of sample remaining in the junction between adjacent processingstations during an incubation period, while still allowing sample flowthrough the thermal insulator when a higher pressure is applied to thesample in an adjacent processing station. This design simplifies theoperation of multiple thermal insulators.

In an alternative embodiment, the processing stations can be spacedapart to inhibit conductive heat transfer between adjacent processingstations and, thereby, eliminate the need for insulators between thestations.

Operation of the thermal cycling reaction unit 124 will be generallydescribed with reference to FIGS. 13A-13G. The thermal cycling processbegins by opening each of the processing stations, e.g. first processingstation 150A, second processing station 150B, and third processingstation 150C, to receive the sample vessel within the opening 114, asillustrated in FIG. 13A. After the sample vessel is loaded withpretreated sample material, or the pretreated sample is dispensed frompretreatment unit 122 into the reaction unit 124, the second processingstation 150B and the third processing station 150C are closed by movingthe compression member 22B and the compression member 22C of eachstation toward the respective stationary member 26B and 26C, asillustrated in FIG. 13B. As the second processing station 150B and thethird processing station 150C are closed, the sample vessel iscompressed between the compression member and the stationary member,displacing the sample within the sample vessel into a segment of thesample vessel adjacent the first processing station 150A.

Next, the compression member 22A and the insulator 146A can compress thesample vessel to adjust the sample volume contained within the segmentof the sample vessel adjacent the first processing station 150A, as wellas the surface area to volume ratio of the segment. The insulator 146Acan then be closed to seal the sample in the first processing station150A, as illustrated in FIG. 13C. Alternatively, if the device 100 isprovided with a sample pretreatment unit, the sample pretreatment unitcan function to close the sample vessel within the first processingstation 150A. Other alternatives include pre-sealing the sample vesselafter loading a sample, or providing the sample vessel with pressuregates, discussed below, formed between adjacent reaction zones. Once thesample is sealed within the first processing station 150A, the samplecan be heated or cooled by the first processing station 150A. In PCRthermal cycling, for example, the sample can be heated to a temperatureeffective to denature the nucleic acid sample. Preferably, the samplevessel is pressed into contact with the compression member 22A and thestationary member 26A by the compression member 22A to flatten thesample vessel and to ensure good thermal contact between the samplevessel and the compression member 22A and the stationary member 26A. Thecompression member 22A can also optionally periodically squeeze thesample vessel to agitate the sample and to generate sample flow in thesegment of the sample vessel during the reaction period to speed up thereaction.

After a predetermined period, the second processing station 150B can beopened to allow the sample to move into the second processing station150B, as illustrated in FIG. 13D. Next, the first processing station150A closes, compressing the sample vessel and moving the entire sample,within the vessel 16, into a segment of the sample vessel adjacent thesecond processing station 150B, as illustrated in FIG. 13E. The thirdprocessing station 150C can then open to allow the sample to move intothe segment of the sample vessel adjacent the third processing station150C, as illustrated in FIG. 13F. The second processing station 150Bcloses, compressing the sample vessel and moving the sample completelyinto the segment of the sample vessel adjacent the third processingstation 150C, as illustrated in FIG. 13G. The sample can then be heatedor cooled by the third processing station 150C for a set time period. InPCR thermal cycling for example, the sample can be heated to atemperature effective to anneal the nucleic acid sample in the thirdprocessing station 150C. The heat sink 166 can facilitate thetemperature transition from the denaturing temperature of the firstprocessing station 150A to the annealing temperature of the thirdprocessing station 150C by dissipating excess heat to the environment.Thus, the sample can be moved from the denaturing step at the firstprocessing station to the annealing step at the third processingstation.

After a predetermined time period, the second processing station 150Bopens to allow the sample to move into the second processing station, asillustrated in FIG. 13F. The third processing station 150C then closes,compressing the sample vessel 16 and moving the sample entirely into thesegment of the sample vessel adjacent the second processing station150B, as illustrated in FIG. 13E. The sample can then be heated orcooled by the second processing station 150B for a set time period. InPCR thermal cycling for example, the sample can be heated to atemperature effective to elongate the nucleic acid sample. Uponconclusion of the elongation step, the sample can be returned to thesegment of the sample vessel adjacent the first processing station 150Ato repeat the cycle, i.e., denaturing and annealing and elongating or,the sample can be moved to a segment of the sample vessel adjacent thesample detection unit 126 if PCR thermal cycling is completed.

The illustrated thermal cycling reaction unit 124 provides denaturing inthe first processing station 150A, annealing in the third processingstation 150C, and elongation in the second processing station 150B, asthis arrangement is deemed thermodynamically efficient. One skilled inthe art will appreciate, however, that the PCR thermal cycling steps canoccur in any of the processing stations without departing from the scopeof the present invention.

Sample thermal cycling using the reaction unit 124 of the presentinvention results in faster thermal cycling times and lower energyconsumption compared to conventional thermal cycling devices. Samplevessel shape alteration, i.e. flattening, by the reaction unit 124results in significant increases in the surface/volume ratio and samplevessel contact with the members of the reaction unit. This allows theprocessing stations of the reaction unit 124 to heat the sample moredirectly, increasing the sample temperature ramping rate and avoidingunnecessary temperature ramping of the members and thus decreasing theamount of energy consumed. Equally important is that sample vessel shapealteration provides for the uniform transfer of thermal energy to thesample, dramatically reducing reaction mixture temperature gradients.The reaction unit 124 further allows the use of fluid flow to mix thesample as the sample is moved from one processing station to another.

Moreover, the reaction unit 124 allows the use of a disposable,single-use sample vessel that minimizes contamination of the sample,contamination of the reaction unit and exposure of the operator tobiohazards. Additionally, the reaction unit 124 does not require a fluidhandling system, as the sample can be moved within the sample vesselduring processing.

Referring again to FIG. 7, the reaction unit 124 can optionally includea reaction sensor 168 for monitoring the reaction in real-time withinthe reaction unit 124 by analyzing the sample, including any reactionproducts from the reaction with the sample. The reaction sensor 168 caninclude an integral light source 169 for applying excitation energy tothe sample within the sample vessel. Alternatively, a light source, orother source of excitation energy, can be provided separate from thereaction sensor 168. The reaction sensor 168 can be an optical sensorfor measuring light, for example fluorescent light, emitted from thesample or from fluorescent probes within the sample. In the case of PCR,any known real-time PCR detection system can be employed, including, forexample, using fluorescent dyes, such as ethidium bromide, intercalatinginto the DNA molecule, using a dual labeled probe tagged with a reporteddye and a quenching dye, or using hybridization probes, which willresult in Fluorescence Resonance Energy Transfer (FRET) only when thetwo probes are hybridized and in close proximity. In each of theseapproaches, the fluorescence signal is substantially proportional to theamount of specific DNA product amplified. The reaction detection sensor168 is placed to monitor the fluorescence from the sample, preferably inthe annealing processing station, or other processing stations of thereaction unit, dependent on the assay selected. Multiple sensors or aspectrum sensor can be used when detection of multiple wavelength lightis required. The detected signal is then sent to the CPU 174 for furtheranalyzing the amount of product.

Continuing to refer to FIG. 7, the sample detection or analysis unit 126of the device 100 is provided to analyze the sample after processing bythe reaction unit 124. The analysis unit 126 is preferably positionedproximate the reaction unit 124. In one embodiment of the invention, asource of excitation energy, for example a light source, can applyexcitation energy to the sample and a signal detector, for example anoptical sensor, can detect light emitted from the sample in response toillumination by the excitation light. Specific illustrative practices,include detecting the transmission of light through the sample,detecting reflected light, detecting scattering light, and detectingemitted light. The detected light, in the form of the signal output fromthe sensor, can be analyzed by a CPU 174 provided in the device throughknown signal processing algorithms. Suitable sample analysis systems,employing a light source and an optical sensor or sensors, detectssignals including light intensity at a given wavelength, phase orspectrum of the light, as well as location of the signal. For example,the flow induced testing system described in U.S. patent applicationSer. No. 09/339,056 and the multi-layer testing system described in U.S.patent application Ser. No. 09/339,055, both of which are incorporatedherein by reference, describe suitable sample analysis systems.

In the case of a PCR based assay, gel electrophoresis or capillaryelectrophoresis can be employed to analyze the nucleic acid sample, asillustrated in FIGS. 7 and 14. Suitable nucleic acid sizing gels includeagarose and polyacrylamide. The gel 184 can be introduced to the samplevessel 16 during processing or, preferably, is pre-loaded into ananalysis segment 210 of the sample vessel, as discussed in more detailbelow. The exemplary analysis unit 126 includes a light source 170 forilluminating the nucleic acid sample and the gel and an optical sensor172 in the form of linear charged couple device (CCD). Electrodeactivators 176 operate to insert a positive electrode 180 and a negativeelectrode 182 into the sample vessel 16. The positive electrode 180 andthe negative electrode 182 are electrically connected to a voltagesource, which creates a voltage difference between the electrodes. Asnucleic acid products are negatively charged, the nucleic acid productswithin the sample will move through the gel 184 toward the positiveelectrode 180. The gel separates the sample components by size, allowingsmaller components, such as nucleic acid products, to travel faster, andthus, further, than larger components. A suitable dye or fluorescent tagcan be introduced to gel to identify the nucleic acid products. Lightfrom the light source 170 can illuminate the dyed or tagged nucleic acidproducts in the gel and the optical sensor 172 can then identify theilluminated nucleic acid products. The output signal of the opticalsensor 172 can be analyzed by CPU 174 according to known signalprocessing method to determine the presence, absence, quantity or othercondition of the nucleic acid sample.

Alternatively, the nucleic acid sample can be analyzed in accordancewith conventional nucleic acid analysis methods, such as, for example,chemiluminescence, fluorescently labeled primers, antibody capture, DNAchip, and/or magnetic bead detection methods.

One skilled in the art will appreciate that the processing units and theprocessing stations of the above-described exemplary embodiments of thesample processing device of the present invention can be arranged in anyorder depending on the sample being processed and the process beingutilized. The sample processing device of the present invention mayinclude any combination of the processing units and processing stationsdescribed herein, as well as additional processing units and processingstations that will be apparent to those skilled in the art upon readingthis disclosure. Moreover, the sample processing device may include onlya single processing unit, such as, for example, a reaction unit forthermal cycling a sample, or may include a only a single processingstation, such as, for example, a processing station for displacing aspecified volume of reagent or sample.

FIGS. 8-12 illustrate alternative embodiments of a reaction unit 250 forthermal cycling a sample according to the present invention. Thereaction unit 250 can include one or more openings 252 for receiving oneor more sample vessels 16. The embodiments illustrated in FIGS. 8-12have three openings 252, permitting the simultaneous thermal cycling ofup to three samples. The reaction unit 250 comprises three processingstations: a first processing station 254, a second processing station256, and a third processing station 258. Thermal insulators 260A-260Dare positioned between the processing stations and at the top of thefirst processing station 254 and the bottom of the third processingstation 258.

Referring specifically to FIGS. 8 and 10, the first processing station254, as well as the second and third processing stations 256 and 258,includes an embedded heat element 262 for transferring thermal energy tothe sample vessel when the sample vessel is positioned within an opening252. The heat element 262 can be a Kapton heater, a Nomex heater, a Micaheater, a silicone rubber heater or any other thermal energy transferelement suitable for delivering thermal energy. The heat element 262 canbe seated in a recess 264 formed in the processing station 254 andsecured to the processing station by an adhesive or other attachmentmeans. The heat element 262 of each of the processing stations ispreferably coupled to a temperature controller 266 for controlling thetemperature of the heat element. One or more temperature sensors 268 canbe positioned in the processing station 254 to measure the temperatureof the processing station 254. The temperature sensor 268 can be coupledto the thermal controller 266 such that the temperature controller 266can monitor and adjust the temperature of the processing station in afeedback control manner.

Referring to FIGS. 10 and 11, each processing station comprises astationary member 270 and a compression member 272 adapted to compressthe sample vessel selectively within one or more of the openings 252 andthereby move the sample within the sample vessel. The compression member272 is preferably complimentary in shape to the stationary member 270and includes a plurality of finger-like closure elements or shutters 274sized and shaped to slide within the openings 252. Guide rails 276 arepositioned on either side of the compression member. The guide rails 276are preferably sized and shaped to fit within grooves 278 formed in theside walls of the stationary member 270. The combination of the guiderails 276 and the grooves 280 allow the compression member 272 toreciprocate relative to the stationary member 270 to selectively openand close the openings 252.

Each thermal insulator 260 can be configured in a manner analogous tothe processing stations. For example, the thermal insulator 260Bcomprises an insulator stationary member 280 and an insulatorcompression member 282 adapted to compress a sample vessel within one ormore of the openings 252. The insulator compression member 282 includesa plurality of finger-like closure elements or shutters 284 sized andshape to slide within the openings 252 to selectively open and close theopenings 252.

Each compression member 272 and insulator compression member 282 iscoupled to a driver, such as an electromagnetic driver mechanism, asdescribed above, or any other mechanism for imparting motion, preferablyreciprocating motion, to the compression members. Each compressionmember can be coupled to an arm 286 for providing a connection betweenthe compression member and the driver, as best illustrated in FIGS.9-11. In one embodiment, illustrated in FIG. 10, the arms 286 are hollowtubes that receive coiled springs 288 and dowels 290. The springs 288operate to bias the compression members 272, 282 in a direction awayfrom the stationary member 270 and the insulator stationary member 280,respectively. An elastic element, such as the coiled spring used here,provides a simple mechanism for assisting the driver to regulate thecompressing pressure applied to the sample vessel. The driver can be amotor 292 for driving a rotating shaft, as best illustrated in FIG. 8.The rotary motion of the shaft can be translated to reciprocating motionthrough cams 294 provided for each of the compression members 272 and282. The cams 294 are coupled to the arms 286. The cams 294 can beconfigured to selectively open and close the compression members 272 and282 in accordance with conventional cam design methods.

In one alternative embodiment of the reaction unit, the compressionmembers 272 and 282 of each of the processing stations and insulatorsinclude holes 296 for receiving a cam 294 and a linear spring element298. Spring elements 298 each operate to bias a respective compressionmember in a direction away from the corresponding stationary member. Thecams 294, in combination with the springs 298, act to impartreciprocating motion to the actuators and regulate the compressingpressure on the sample vessel.

FIGS. 19A-19C illustrate a further embodiment of the reaction unit ofthe present invention. The reaction unit 350 includes nine openings 352for receiving up to nine sample vessels simultaneously. The reactionunit 350 includes three processing stations: a first processing station354, a second processing station 356, and a third processing station358. Thermal insulators 360A-360D are positioned adjacent each of theprocessing stations and at the top of the first processing station 354and the bottom of the third processing station. Top thermal insulator360A and bottom thermal insulator 360D are movable independent of thefirst processing station 354 and the third processing station 358,respectively. Intermediate thermal insulators 360B and 360C are coupledto the first processing station 354 and the second processing station356, respectively.

Each processing station comprises a stationary member 370 and acomplementary compression member 372 adapted to compress the samplevessel selectively within one or more of the openings 352 and therebymove the sample within the sample vessel. Each stationary member 370 hasa projection 374 aligned with one of the openings 352. The compressionmembers 372 are each provided with a projection 376, positioned on anopposite side of the opening 352. When a compression member 372 is slidon the corresponding stationary member 370, the projections 374 and 376engage and close the openings 352 therebetween.

Each compression member 372, as well as intermediate thermal insulators360B and 360C, include an arm 380 coupled by a cam 384 to a rotary shaft382. A stationary insulator member 362 is coupled, and aligned with anedge of each opening 352 on each stationary member 370. Each stationaryinsulator member 362 is inserted in each of the openings of a movableinsulator compression member 360 to react to compression and open orclose the opening. The shaft 382 is rotated by a stepper motor or aservo motor 386. The cams 384 translate the rotation of the shaft 382into linear reciprocal motion, which is imparted to the arms 380 toaffect selective opening and closing of the openings 352 and compressionof the sample vessels therein.

Each arm 380 includes an inner shaft 390 received within an outer sleeve392. A spring 394 is interposed between the inner shaft 390 and therespective compression member or thermal insulator. A second spring 396is positioned on an opposite side of the respective compression memberor thermal insulator. The spring 394 cooperates with the second spring396 to allow the compression member or thermal insulator to “float”along the axis of the arm 380. In this manner, the arm 380 can applysufficient force to the compression member or thermal insulator tocompress the sample vessel within an opening 352 and, thereby, displacesubstantially all of the sample from the compressed portion of thesample vessel. An increase of pressure within the sample vessel, forexample, from the compression of an adjacent portion of the samplevessel, however, can cause the sample to displace within the samplevessel through the compressed portion of the sample vessel, as thesprings 394 and 396 will allow small axial movements of the compressionmember or thermal insulator.

Each stationary member 370 and compression member 372 can be providedwith an embedded thermal energy transfer device 398 for each opening 352to apply thermal energy to the sample vessel within the opening 352. Inaddition, the stationary member 370 and compression member 372 caninclude temperature sensors 399 associated with each energy transferdevice 398 to monitor the temperature of the sample vessel.

FIGS. 15A and 15B illustrate embodiments of a sample vessel 16 accordingto the present invention. The illustrated sample vessel 16 is a closedtubule system that provides a disposable, single use container andreaction vessel for the sample. The sample vessel 16 can be constructedof a resiliently compressible, flexible, and ultra-high strengthmaterial, such as polyethylene or polyurethane. The sample vessel 16 canhave a seamless, flattenable cross-sectional profile and thin-walledconstruction that is optimized for fast and uniform heat transfer, formaximum surface contact with the sample, and for high pressureresistance. Preferably, the walls are constructed to converge when thesample vessel is compressed in a direction perpendicular to thelongitudinal axis of the sample vessel such that the volume of thecompressed portion of the sample vessel decreases and the ratio of thesurface area to the volume of the compressed portion increases, withoutfracturing of the sample vessel. In one illustrative preferred practice,the walls of the sample vessel 16 have a wall thickness of approximately0.01 mm to 0.5 mm. Experimental results indicate that constructing asample vessel having a wall thickness within this preferred rangesignificantly increases the efficiency of heat transfer to the sample.In an alternative embodiment, a two-layer wall structure can be used,with the inner layer providing bio-compatibility, using material such aspolyethylene or polyurethane, and the outer layer providing lowerpermeability, using material such as high density polyethylene oraluminum foil. In addition, the material selected to construct theportions of the wall of the sample vessel, such as a detection segmentof the sample vessel 16, can be optically transmissive over a selectedwavelength range to facilitate optical analysis of the sample within thesample vessel.

The sample vessel 16 can be divided into multiple segments by one ormore pressure gates 32. In the case of PCR testing, for example, thesample vessel can be divided into a sample collection segment 205, asample pretreatment segment 206, a sample reaction segment 208, and asample analysis segment 210. The illustrated pressure gates 32 areinternal to the tubule structure of the vessel 16 and provide a fluidtight seal between the segments of the sample vessel 16, under normaloperating conditions. Preferably, the pressure gates 32 open upon theapplication of pressure greater than a certain value, for example,approximately 3 atmospheres. When external pressure is provided to onesegment, the pressure gate 32 can open, allowing the sample to flow fromthe high pressure compartment to the low pressure compartment.

The sample vessel 16 can include a handling portion having a generallyrigid construction to facilitate handling of the sample vessel. Thehandling portion can be coupled to one or more of the segments of thesample vessels used to contain the sample. For example, the handlingportion can be a cylindrical sleeve constructed of a generally rigidmaterial, such as a plastic or a metal, that is sized and shaped to fitover one or more of the segment of the sample vessel. In one embodiment,the cylindrical sleeve can be removable and replaceable. Alternatively,the handling portion can be a rigid segment, such as a rigid ring,positioned at an end of the sample vessel or between two segments of thesample vessel. In the embodiments illustrated FIGS. 15A and 15B, thehandling portion is a segment of the sample vessel having an increasedwall thickness. For example, the sample collection segment 205 and thesample pretreatment segment 206 have a wall thickness greater than thewall thickness of the reaction segment 208. The increased wall thicknessprovides sufficient rigidity to the sample collection segment 205 andthe sample pretreatment segment 206 to facilitate handling of the samplevessel 16. In one embodiment, the wall thickness of the handling portionis greater than 0.3 mm.

The sample vessel 16 can include an instrument, such as a samplingpipette or a needle 107, for direct collection of the sample to betreated and analyzed within the sample vessel 16, as illustrated in FIG.15A. The needle 207 can be positioned at one end of the sample vessel 16and can be connected to the sample collection chamber 205 through aconduit 209 formed in the wall of the sample vessel 16. A needle cover211 can be provided to secure the needle 207 prior to and after use. Theneedle cover 211 can be, for example, a penetrable rubber cover or aremovable plastic cover.

In another embodiment, illustrated in FIG. 15B, a sampling instrument214, such as a pipette, a stick, or a tweezer, can be coupled to a cover212 that selectively closes the conduit or opening 209 formed in thewall of the sample vessel. The cover 212 can include a reservoir 216 forcontaining a reagent and a sample during sampling. For sampling, thecover 212 can be removed from the sample vessel to expose the samplinginstrument 214. The sampling instrument 214 can be used to collect thesample, by pipetting, swabbing, or gathering the sample, for example,and then the sampling instrument 214 can be inserted into the samplecollection segment 205 through the conduit 209. The sample can then beintroduced to the sample collection segment 205 by compressing the cover216 to displace the sample from the reservoir 216. Alternatively, thesample can be introduced to the sample collection segment 205 or toanother segment of the sample vessel, depending of the segments presentin the sample vessel, after collection by a separate instrument.

Sample vessel 16 can be particularly suited for PCR testing using thesample processing device of the present invention, as described above.For example, nucleic acid extraction can be performed within the samplepretreatment segment 206 of such a sample vessel 16. A cell lysesreagent, for example, GeneReleaser™ from Bioventures, Release-IT fromCPG Biotech, or Lyse-N-Go™ from Pierce, or other extraction reagents canbe introduced to the pretreatment segment 206 to extract nucleic acidfrom the initial sample. Extraction reagents can be stored within thepretreatment segment 206 or can be delivered to the segment.Additionally, one or more filters can be positioned within thepretreatment segment 206 of the sample vessel to separate nucleic acidfrom unwanted cellular debris.

After incubation of the sample for certain time period, a portion ofpretreated sample can be moved into the reaction segment 208. For areaction sample volume of approximately 5 μl-25 μl, a PCR reactionsegment 208 of the sample vessel 16 according to one illustrativepractice of the invention has a wall thickness, indicated by referencecharacter t in FIG. 15A, of approximately 0.01 mm-0.3 mm, a diameter ofless than approximately 6 mm, and a length of less than approximately 30mm. PCR reagents, such as nucleotides, oligonucleotides, primers andenzymes, can be pre-packaged in the reaction segment or reactionsegments 206, or can be delivered, for example, through the walls of thesample vessel using a needle, using for example, a reagent injectorcartridge described below, before moving the sample into the segment.

A pre-packaged reagent storage segment 214 can be used to stored apre-packaged reagent. Such a reagent storage segment can be formedbetween any two adjacent processing segments and may store any reagentneeded for a reaction. For example, the reagent storage section 214 canstore PCR reagents, while reagent storage sections 236 and 244,described below, may include detection reagents. If the reagent storagesegment 214 is utilized, the sample vessel 16 can be compressed at thereagent storage segment 214 to displace the reagent into thepretreatment segment 206. Alternatively, the sample can be moved fromthe pretreatment segment 206, through the reagent storage segment 214where mixing with the reagent, to the reaction segment 208.

A self-sealing injection channel 218 can be formed in the sample vesselto facilitate delivery of reagent or other materials to the samplevessel, as illustrated in FIG. 16. The illustrated self sealinginjection channel 218 is normally substantially free of fluidic materialand is capable of fluid communication with the adjacent segment in thevessel. An injection of reagent through an injection channel occurspreferably prior to moving any sample into the segment to avoidcontamination. In addition, the sample treatment devices of theinvention can utilize a reaction cartridge 220 with a single or multipleneedles 222 in fluid communication with one or more reservoirs, asillustrated in FIG. 17. The reaction cartridge 220 can be used to injector deposit reagent or other materials, simultaneously, or sequentiallyinto multiple segments of the sample vessel. Suitable self-sealinginjection channels and reagent cartridges are described in U.S. patentapplication Ser. No. 09/339,056, incorporated herein by reference.

One skilled in the art will appreciate that while it may be preferablefor the wall of the sample vessel to uniform along the circumference andthe longitudinal axis of the vessel, only a portion of the wall alongthe circumference and/or the longitudinal axis of the vessel need beresilient and compressible and have the preferred thickness to affectflattening of the sample vessel. Thus, the sample vessel need not have auniform axial or circumferential cross-section.

PCR thermal cycling can be performed in the reaction segment 208 of thesample vessel 16. The thin walled, compressible construction of thesample vessel 16 greatly improves the rate and efficiency of thermalcycling. The construction of the sample vessel allows the vessel todeform or flatten readily, increasing thermal contact with the reactionunit of the device 10 and increasing surface/volume ratio of the samplewithin the sample vessel. As a result, the reaction mixture ramping rateis increased and thermal energy is more uniformly transferred to thesample.

PCR analysis can be performed in the sample vessel 16. For example,real-time detection methods can be used within the reaction segment 208;gel electrophoresis or other nucleic acid detection methods can be usedwithin the analysis segment 210 to analyze the sample. In the case ofgel electrophoresis, a gel can be introduced to the analysis segment 210to facilitate gel electrophoresis, as described above in connection withFIG. 14.

In one preferred embodiment, illustrated in FIG. 15A, the analysissegment 210 is divided into two electrophoresis capillaries, namely, asample capillary 230 and a control capillary 232, by adiametrically-central divider 234. Pressure gates 32 at either end ofthe capillaries control the movement of the sample and the reagents intoboth capillaries. Each capillary is filled with an electrophoresis gelsuch that gel electrophoresis can be performed simultaneously in bothcapillaries. A pair of electrodes 240, for both capillary 230 and 232,can be positioned within the walls of the sample vessel. A reagentstorage segment 236 can be provided at the proximal end of the samplecapillary 230 for storing reagent within the sample vessel prior to thesample entering the sample capillary 230. A control material can bestored in a control storage segment 242 positioned at the proximal endof the control capillary 232. A reagent can be stored in a reagentsegment 244 positioned at the distal end of the capillaries and incommunication with both the sample capillary 230 and the controlcapillary 232 for detection or display signal. The presence of thecontrol capillary 232 facilitates detection and analysis of the sampleby providing a basis of comparison for the sample analysis.

One skilled in the art will appreciate that the number of segmentswithin the sample vessel is dependent upon the sample being processedand the processing methods being employed. For example, in the case ofPCR testing, the number of segments within the sample vessel can bethree or more. Alternatively, thermal cycling and analysis may beperformed in one segment, reducing the number of segments to two. Incertain cases, an isothermal nucleic acid amplification method, forexample, only one segment may be necessary.

FIG. 18 illustrates a sample vessel 416 particularly suited for use in amulti-opening sample processing device such as, for example, the sampleprocessing device illustrated in FIG. 6. The sample vessel 416 includesan opening 420 for receiving the sample, a cap or closure 424 forselectively closing and sealing the opening 420, and a sample containingportion 426 within which the sample can be treated. The opening 420 isformed in a handling portion 428 that is preferably constructed of agenerally rigid or semi-rigid material, such as plastic or metal, tofacilitate handling of the sample vessel 416. The handling portion 428includes a collar 430 against which the cap 424 seats. Sample materialcan be introduced into the sample containing portion 426 of the samplevessel 416 through the opening 420. The collar 428 preferable tapersfrom a larger diameter to the smaller diameter of the sample containingportion 426. The sample containing portion 426 is preferable constructedof a resiliently compressible, flexible, and ultra-high strengthmaterial, such as polyethylene or polyurethane. The sample containingportion 426 can have a seamless, flattenable cross-section profile andthin-walled construction that is optimized for fast and uniform heattransfer, for maximum surface contact with the sample, and for highpressure resistance. In accordance with one embodiment, the samplecontaining portion 426 has a wall thickness of approximately 0.01 mm-0.3mm. Preferably, the sample containing portion 426 of the sample vessel416 is in a flattened state prior to introduction of the sample.Introduction of the sample to the sample containing portion 426 willcause the walls of the sample containing portion to separate and thevolume of the sample containing portion to increase. Compression of aselected portion of the sample containing portion 426 can cause thesample to displace to another portion within the sample containingportion along the length of the sample vessel. The surface of the samplevessel can be chemically treated to reduce a surface effect on thereaction.

The embodiments of the sample vessel described herein in connection withFIGS. 14-16 and 18, are not limited to use with the embodiments of thesample processing device described herein. The sample vessel of thepresent invention may be used with any sample testing or processingsystem. Likewise, the sample processing device of the present inventionis not limited to use with the sample vessels described herein. Othersample vessels may be used without departing from the scope of thepresent invention.

Certain changes may be made in the above constructions without departingfrom the scope of the invention. It is intended that all mattercontained in the above description or shown in the accompanying drawingsbe interpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are to cover allgeneric and specific features of the invention described herein, and allstatements of the scope of the invention which, as a matter of language,might be said to fall therebetween.

Having described the invention, what is claimed as new and desired to besecured by Letters Patent is:
 1. A thermal cycler comprising aprocessing unit having an opening to receive a sample vessel containinga sample, the processing unit having a first processing station, asecond processing station, and a third processing station positionalalong the opening, the first processing station including a firstcompression member adapted to compress the sample vessel within theopening and a first energy transfer element for transferring energy tothe sample at the first processing station, the second processingstation including a second compression member adapted to compress thesample vessel within the opening and a second energy transfer elementfor transferring energy to the sample at the second processing station,and the third processing station including a third compression memberadapted to compress the sample vessel within the opening and a thirdenergy transfer element for transferring energy to the sample at thethird processing station, wherein compression of the sample vessel by ofone of the compression members displaces the sample within the samplevessel between the processing stations.
 2. The thermal cycler of claim1, further comprising at least one sensor for detecting a signal from tocontent within the sample vessel.
 3. The thermal cycler of claim 2,wherein the sensor comprises an optical sensor for measuring lightsignal from the contents with the sample vessel.
 4. The thermal cyclerof claim 3, wherein the light signal comprises fluorescent light.
 5. Thethermal cycler of claim 2, wherein the sensor monitors the signal fromthe content within the sample vessel in real time.
 6. A thermal cyclercomprising a processing unit having an opening to receive a samplevessel containing a sample, the processing unit having a firstprocessing station and a second processing station positioned along theopening, the first processing station including a first compressionmember adapted to compress the sample vessel within the opening and afirst energy transfer element for transferring energy to the sample atthe first processing station, and the second processing stationincluding a second compression member adapted to compress the samplevessel within the opening and a second energy transfer element fortransferring energy to the sample at the second processing station,wherein compression of the sample vessel by of one of the compressionmembers displaces the sample within the sample vessel between theprocessing stations.
 7. The thermal cycler of claim 6, furthercomprising ax least one sensor for detecting a signal from the contentwithin the sample vessel.
 8. The thermal cycler of claim 7, wherein thesensor comprises an optical sensor for measuring light signal from thecontents with the sample vessel.
 9. The thermal cycler of claim 8,wherein the light signal comprises fluorescent light.
 10. The thermalcycler of claim 7, wherein the sensor monitors the signal from thecontent within the sample vessel in real time.
 11. The thermal cycler ofclaim 1, further comprising at least one energy insulator positionedadjacent at least one processing station.
 12. The thermal cycler ofclaim 1, wherein at least one of the the energy transfer elementscomprises at least one of an electronic heat element, a microwavesource, a light source, an ultrasonic source and a cooling element. 13.The thermal cycler of claim 1, further comprising a control systemcoupled to at least one energy transfer element to control the energytransferred to or from that energy transfer element.
 14. The thermalcycler of claim 13, further comprising a temperature sensor coupled tothe control system.
 15. The thermal cycler of claim 1, wherein at leastone processing station further comprises a heat sink.
 16. The thermalcycler of claim 1, wherein at least one processing station includes astationary member opposing the respective compression member across theopening, wherein the respective compression member compresses the samplevessel against the stationary member within the opening.
 17. The thermalcycler of claim 1, further comprising a driver coupled to at least onecompression member to selectively move that compression member andthereby compress the sample vessel within the opening.
 18. The thermalcycler of claim 17, wherein the driver is a motor and is coupled to theat least one compression member by a cam.
 19. The thermal cycler ofclaim 17, wherein the driver is an electromagnetic actuating mechanism.20. The thermal cycler of claim 1, further comprising an energy sourcefor applying energy to at the sample within the sample vessel togenerate a signal from the sample.
 21. The thermal cycler of claim 1,further comprising an electrophoresis system comprising a pair ofelectrodes adapted to have a predetermined voltage difference and anelectrode actuator for inserting the electrodes into the sample vessel.22. The thermal cycler of claim 1, further comprising a reagent injectorcartridge actuator adapted to receive a reagent injector cartridgehaving at least one needle in fluid communication with a reagentreservoir, the reagent injector cartridge actuator operable to move thereagent injector cartridge to inject a quantity of reagent into thesample vessel.
 23. The thermal cycler of claim 1, wherein compression ofthe sample vessel by one of the compression members displaces a reagentwithin the sample vessel between the processing stations.
 24. A methodof thermal cycling, comprising: adding a sample to a sample vessel;introducing the sample vessel into a thermal cycler as set forth inclaim 1; compressing the sample vessel with the first compression memberto move the sample within the sample vessel from the first processingstation to the second processing station; transferring energy to thesample at the second processing station; compressing the sample vesselwith the second compression member; and transferring energy to thesample at the first processing station.
 25. The method of claim 24,further comprising adding a reagent to the sample in the sample vessel.26. The method of claim 24, further comprising heating the sample in thefirst processing unit to a first temperature.
 27. The method of claim26, further comprising heating the sample in the second processing unitto a second temperature.
 28. The method of claim 27, wherein the firsttemperature is effective to denature nucleic acid in the sample and thesecond temperature is one at which nucleic acid annealing and nucleicacid synthesis can occur.
 29. The method of claim 24, further comprisinganalyzing the sample by detecting a signal from the sample, andanalyzing the detected signal to determine a condition of the sample.30. The method of claim 29, wherein analyzing further comprises applyingan excitation energy to the sample.
 31. The method of claim 24, furthercomprising conducting electrophoresis analysis of the sample by:applying a selective voltage to the sample; detecting light emitted fromthe sample; and analyzing the detected light to determine a condition ofthe sample.
 32. The method of claim 24, further comprising: applying anexcitation energy to a bio-array member contained within the samplevessel; detecting light emitted from the bio-array member; and analyzingthe detected light to determine a condition of the sample.
 33. Themethod of claim 24, further comprising agitating the sample within thesample vessel.
 34. A method of thermal cycling, comprising: adding asample to a sample vessel; introducing the sample vessel into a thermalcycler as set forth in claim 1; compressing the sample vessel with thefirst compression member; transferring energy to the sample with thesecond energy transfer element; compressing the sample vessel with thesecond compression member; transferring energy to the sample with thethird energy transfer element; compressing the sample vessel with thethird compression member; and transferring energy to the sample with thefirst energy transfer element.
 35. The method of claim 34, furthercomprising agitating the sample within the sample vessel.
 36. The methodof claim 34, further comprising heating the sample in the firstprocessing unit to a first temperature.
 37. The method of claim 36,further comprising heating the sample in the second processing unit to asecond temperature.
 38. The method of claim 37, further comprisingheating the sample in the third processing unit to a third temperature.39. The method of claim 38, wherein the first temperature is effectiveto denature nucleic acid in the sample, the second temperature is one atwhich nucleic acid annealing can occur, and the third temperature is oneat which nucleic acid synthesis can occur.
 40. A method of thermalcycling, comprising: adding a sample to a sample vessel; introducing thesample vessel into a thermal cycler as set forth in claim 6; compressingthe sample vessel with the first compression member to move the samplewithin the sample vessel from the first processing station to the secondprocessing station; transferring energy to the sample at the secondprocessing station; compressing the sample vessel with the secondcompression member; and transferring energy to the sample at the firstprocessing station.
 41. The method of claim 40, further comprisingadding a reagent to the sample in the sample vessel.
 42. The method ofclaim 41, further comprising heating the sample in the first processingunit to a first temperature.
 43. The method of claim 42, furthercomprising heating the sample in the second processing unit to a secondtemperature.
 44. The method of claim 43, wherein the first temperatureis effective to denature nucleic acid in the sample and the secondtemperature is one at which nucleic acid annealing and nucleic acidsynthesis can occur.
 45. The method of claim 40, further comprisinganalyzing the sample by detecting a signal from the sample, andanalyzing the detected signal to determine a condition of the sample.46. The method of claim 45, wherein analyzing further comprises applyingan excitation energy to the sample.
 47. The method of claim 40, furthercomprising conducting electrophoresis analysis of the sample by:applying a selective voltage to the sample; detecting light emitted fromthe sample; and analyzing the detected light to determine a condition ofthe sample.
 48. The method of claim 40, further comprising: applying anexcitation energy to a bio-array member contained within the samplevessel; detecting light emitted from the bio-array member; and analyzingthe detected light to determine a condition of the sample.
 49. Themethod of claim 40, further comprising agitating the sample within thesample vessel.